Peptides and proteins for desensitizing subjects allergic to bee venom and compositions containing same

ABSTRACT

The invention concerns peptides and proteins for desensitizing specifically the vast majority of subjects allergic to bee venom and compositions containing said peptides or proteins. The peptides are selected in the group consisting of: the fragment (1) corresponding to positions P85-97 of the major bee venom allergen, the fragment (2) corresponding to positions P81-93 of the major bee venom allergen, the fragment (3) corresponding to positions P94-106 of the major bee venom allergen, the fragment (4) corresponding to positions P76-88 of the major bee venom allergen, the fragment (5) corresponding to positions P77-94 of the major bee venom allergen, and the fragment (6) corresponding to positions P122-134 of the major bee venom allergen, fragments (I) and (2) forming group (1); fragment (3) forming group (II); fragments (4) and (4) forming group (III) and fragment (6) forming group IV and the mutated fragments of said fragments (1) to (6) which have a binding activity with MHC class (II) molecules identical or higher than those of said fragments (1) to (6). The invention also concerns compositions containing said peptides or proteins.

The present invention relates to peptides and proteins capable ofdesensitizing, in a specific manner, the great majority of subjectsallergic to bee venom, and to compositions containing said peptides orproteins.

Immediate allergy to Hymenoptera (bee, wasp, hornet) affects 15 to 20%of the population, if prick tests are taken into account, but only 0.1to 0.5% of the population is exposed to an anaphylactic-type accident(1, 2). This allergy is characterized by varying manifestations rangingfrom local swelling to systemic reactions such as urticaria, angioedemaor anaphylactic shock. Given the suddenness of the stings,desensitization (specific immunotherapy or SIT) with venoms constitutesthe preferred treatment for this allergy but is not without danger.Indeed, 13% of patients desensitized with bee venom and 5% in the caseof wasp venom are victims of side effects (3). The search for a bettertolerated and equally effective SIT is therefore necessary, inparticular for bee venom.

The speed of the reactions observed in patients allergic to bee venom ischaracteristic of an immediate-type allergy which is mediated by IgEsspecific for the constituents of the venom. The complex mechanism ofthese reactions is summarized below.

IgEs appear gradually under the repeated action of stings and before anysymptom becomes apparent. Although the bee venom comprises numerouspeptides and proteins, all the components do not appear to be allergenic(4). Melittin, for example, induces IgEs in only 30% of patients,whereas the proportion increases to more than 90% for phospholipase A2(PLA2) which is, as a result, considered to be the major allergen (APIm1). The protein sequence of bee venom phospholipase A2 (API m1) isillustrated in FIG. 1 (SEQ ID NO: 8); this sequence is deduced from thatof the complementary cDNA (36).

IgEs possess the property of binding, via their Fc fragment, toreceptors situated on the tissue mastocytes and the blood basophils.When the allergen forms a complex with the specific IgEs bound to themembrane of the basophils or mastocytes, it causes degranulation of thecells and the release of molecules which are responsible for theprincipal manifestations observed during an allergic accident. IgEs arenot solely responsible for the allergy because although the IgE level isan indicator for the disease, it has no diagnostic value for the stateof the patients. It is not rare for patients to have high IgE levelswithout showing symptoms. The appearance of IgEs in allergic patientsresults from the production of type TH2 cytokines such as IL-4, IL-5 andIL-13 and is inhibited by the synthesis of IFN-γ (6).

It is mainly the CD4⁺T lymphocytes which produce these cytokines.Specific T cells which secrete more IL-4 than IFN-γ are effectivelyfound in allergic patients, whereas the T cells isolated fromnonallergic subjects produce more IFN-γ than IL-4.

The TH2-type CD4⁺ T lymphocytes specific for the venom componentstherefore actively participate in the appearance and the maintenance ofthe allergy.

To protect the subjects allergic to bee venom, it was proposed long agoto desensitize the allergic subjects by specific immunotherapy (or SIT).The immunological mechanisms of SIT which are responsible for theimprovement in the patient's condition remain poorly understood. Theinduction of specific IgGs and of an IgG4 subclass (7) as well as thegeneration of suppressive CD8⁺ T cells (8) were initially proposed toaccount for the efficacy of the treatment. However, it was more recentlyobserved that during the desensitization to bee venom, the proliferationof the CD4⁺ T lymphocytes specific for the allergen decreases while thesecretion of IL-4 and IL-5 decreases (9, 10) or is diverted toward theproduction of IFN-γ (11). Very similar results were also obtained withpollen allergens (12). These observations appear to indicate that theimprovement in the patient's condition results from a peripheraltolerance or from a drift toward a TH1-type profile for the CD4⁺ Tlymphocytes specific for the allergen. They are furthermore in agreementwith experiments carried out in animals. It has indeed been shown forseveral antigens that under injection conditions similar to those usedto desensitize (such as subcutaneous injection) the anergy of the Tlymphocytes is induced while a high response is observed for the sameantigens when they are injected in the presence of adjuvant (13, 14).All these experiments make it possible to consider the CD4⁺ Tlymphocytes specific for bee venom as the target cells forimmunotherapy.

The CD4⁺ T lymphocytes possess a rearranged T receptor which allows themto selectively recognize peptide fragments derived from the degradationof the antigen by the presenting cells and presented by the MajorHistocompatibility Complex class II (MHC II) molecules (15). Thedeterminants which these peptide fragments carry and which the Tlymphocytes effectively recognize are called T epitopes.

During desensitization, it is these determinants which are recognized bythe T lymphocytes and which therefore constitute the basic elements forthe production of alternative molecules for specific immunotherapy.

It has indeed been observed in vivo in mice for the allergens Fel d1(cat hair), Der p1 (acarian: Dermatophagoides pterissimus) and Bet v1(birch pollen) that the nasal, oral or subcutaneous administration ofpeptides carrying T epitopes of these allergens inhibits the activationof the specific T lymphocytes (16-18) and modulates the allergicreaction (16, 18).

Ideally, the desensitizing molecules should possess all the epitopes ofthe allergen and be free of reactivity toward the IgEs, so as to avoidthe risks of accidents.

Several types of molecule are already being studied, including in thecontext of bee venom allergy and consist either of peptide fragments orof modified proteins.

Peptide Fragments

Using an empirical approach, the use of peptide fragments (19) todesensitize allergic patients has been proposed on several occasions asan alternative to conventional specific desensitization, including forbee venom allergy (20, 21).

These fragments generally no longer possess reactivity toward IgEs, butthey may also have lost their capacity to be recognized by Tlymphocytes.

More recently, allergen peptide fragments were chosen on the basis oftheir capacity to stimulate T lymphocytes in allergic patients (22).

In the case of the major bee venom allergen (API m1), fragments 50-69and 83-97 have been described as being active during a study comprisinga single patient (23).

In a study comprising forty patients (24), it is fragments 45-62 and81-92 and 113-124 which proved to be active. These three fragments areonly T epitopes for 25 to 45% of the patients and the authors do notexclude the existence of other epitopes (24, 25). These three peptidesare undergoing clinical trial and appear to give encouraging results(22). Muller et al. (22) have used them to desensitize five allergicpatients whose T lymphocytes proliferate in the presence of thesepeptides. No serious systemic effect was observed and the patientsbecame tolerant to bee stings. This demonstrates the benefit of usingpeptides for desensitizing, but does not make it possible to extend theuse of these peptides to other patients.

Another trial using peptides was set up for the cat allergen (Fel d1)(26). Several applications relating to peptides from ragweed (WO93/21321; WO 96/13589), from Japanese cedar pollen (WO 93/01213; WO94/01560) and from ryegrass pollen (WO 94/21675; WO 94/16068) have beenfiled.

All the peptides described in these applications were chosen on thebasis of the stimulation of T lymphocytes in a group of allergicpatients.

The approach followed by these various authors (23, 24 and 30) is basedon cellular tests and not on binding tests. The results observed showthat the active peptides vary according to the patients. In the latterthree studies, the peptides containing the zone 80-90 are those whichare most often a T epitope. They also show that the lymphocytes ofseveral patients are stimulated by peptides containing the C-terminalportion of API m1.

For example, Kämmerer et al. (30) propose, according to the sameprinciple of the stimulation test, using long fragments of API m1, inparticular fragment 90-134. However, these fragments are specific forcertain patients and are not suitable for a significant set of patientsbecause their selection does not take into account the HLA type of thepatients.

The stimulation tests indeed make it possible to select peptidessuitable for desensitizing a given patient (22), but do not make itpossible to extend the use of these peptides to patients other thanthose for which they were produced.

Modified Proteins

Another alternative to the use of native allergens is that of allergensmodified such that they no longer exhibit reactivity toward the IgEsspecific for the allergen, while preserving their reactivity toward theT lymphocytes. As the IgEs are mainly directed against conformationalepitopes, the loss of reactivity can be easily obtained by destroyingthe three-dimensional structure of the allergen, which does not modifyby the T epitopes. That is what was done for mutants of Der f2 (27) andDer p2 (28) in which the disulfide bridges were broken. Another way ofproceeding is to introduce into the allergen point mutations whichaffect the recognition of the IgEs without modifying thethree-dimensional structure (29). The small number of mutationsintroduced is thought not to modify the T epitopes.

The large differences observed between studies indicate theinterindividual variability of the T epitopes and obviously make thechoice of desensitizing molecules difficult. Furthermore, these studies,which use only a group of allergic patients, do not make it possible toensure that all the T epitopes are conserved in the desensitizingmolecule.

Accordingly, the inventors set themselves the objective of providing aset of peptides capable of desensitizing the great majority of subjectsallergic to bee venom. Such a set of peptides has the property of beingeffective in a large number of subjects, whereas the prior art peptidesare active in one allergic subject but may be completely ineffective foranother subject because the latter does not recognize the allergen bythe same determinants.

To do this, the inventors have defined a relationship between thepeptide sequences of the major bee venom allergen (API m1) and MHC classII molecules, both in the alleles of the HLA-DRB1 gene (1st gene), andthe alleles of the DRB3, DRB4 and DRB5 genes (2nd gene), which arepredominant in Caucasian populations. This relationship makes itpossible, unexpectedly, to define molecules for desensitization and forpreventive treatment of allergy to bee venom which takes into accountthe polymorphism of the MHC class II, in particular HLA-DR molecules,and which by virtue of their high specificity, induce betterdesensitization which results in a significantly reduced risk ofaccidents (shocks) during desensitization. This represents an additionaladvantage for the preventive use of such peptides.

The molecules of the major histocompatibility complex (MHC) class II(HLA II in man) are heterodimers expressed on presenting cells andpresent the T epitopes of the antigens to the CD4⁺ T lymphocytes. Thesemolecules are capable of binding a large repertoire of peptides havingvery different sequences, which allows them to present several peptidesper antigen to the T cells.

There are four different types of MHC II molecules per individual (2HLA-DR, 1 HLA-DQ and 1 HLA-DP). The HLA-DR molecule, whose β chain isencoded by the DRB1 gene (1st gene) is the most highly expressed. Therehave currently been recorded more than 200 different alleles for DRB1,which define various antigens or types, as summarized in Table 1 below.

TABLE I Molecules expressed by various HLA-DRB1 alleles Antigen AlleleAlias DR1 DRB1*0101 DR1 DR2 DRB1*1501 DR2w2b DR3 DRB1*0301 DR3w17 DR4DRB1*0401 DR4w4 DRB1*0405 DR4w15 DR7 DRB1*0701 DR7 DR8 DRB1*0802 DR8w2DR9 DRB1*0901 DR9 DR11 DRB1*1101 DR5w11 DR12 DRB1*1201 DR5w12 DR13DRB1*1301 DRB1*1302 DR6w19

Each allele possesses its own binding properties. It therefore binds arepertoire of peptides specific to it and which differs from that foranother allele, even on the same antigen. The broad specificity of theHLA II molecules and the existence of several isoforms and of a highpolymorphism mean that many different fragments of the antigen can bepresented to the T lymphocytes.

The frequencies of each allele (1st gene) are not identical and varyfrom one population to another (35):

the DRB1*1304 allele represents, on its own, 25.4% of the alleles in theSenegalese population against 0% in Germany and in Japan,

the DRB1*0301 allele is observed with a frequency of 10% in theSenegalese and the Germans but only at 0.4% in the Japanese,

in France, only seven alleles exceed 5%. They are the alleles DRB1*0101,DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301 and DRB*1501, asillustrated in Table II below.

TABLE II Frequency of the HLA-DR alleles in several Caucasianpopulations FRA DAN GER ITA ROU SPA US CAN DRB1*0101 9.3 13.0 6.7 6.57.6 6.6 7.3 5.6 DRB1*0301 10.9 10.2 9.4 10.5 11.4 6.7 9.5 12.3 DRB1*04015.6 17.6 8.1 2.3 4.2 5.6 6.7 9.5 DRB1*0701 14.0 14.8 12.3 12.5 8.3 18.914.4 9.4 DRB1*1101 9.2 0.9 9.2 12.4 7.3 1.0 4.4 2.6 DRB1*1301 6.0 8.34.5 4.8 4.4 4.5 5.1 4.7 DRB1*1501 8.0 17.6 7.8 5.6 6.2 9.4 10.3 10.4Total 63 82 58 55 49 53 58 55

They represent, on their own, 63% of the French population. These samealleles are also the most abundant in the other Caucasian populations.Their frequencies vary from 53% (in Spain) to 82% (in Denmark). For theUnited States and Canada, they represent 58 and 55% of the population,respectively. They therefore represent, on their own, 53 to 82% of thealleles in Caucasian populations and are part of the variousspecificities of HLA-DR series.

The 2nd gene encodes the HLA-DRB3, -DRB4 and -DRB5 molecules which areHLA-DR molecules whose β chain is not encoded by the DRB1 gene. Althoughless well known than the molecules derived from the 1st gene, these HLAmolecules are functional and are capable of presenting peptides to the Tlymphocytes (56-59). Their main advantage for the immunotherapy is thatalleles such as DRB3*0101 (9.2%), DRB4*0101 (28.4%) and DRB5*0101 (7.9%)are very frequent in the Caucasian population. They cover, on their own,45% of the gene frequency. They are systematically associated withanother HLA-DR molecule and can therefore complement its specificity. Astrong binding disequilibrium exists between the 1st and 2nd gene, thatis to say that a 2nd gene is very often associated with particularalleles of the 1st gene. The set of DR pseudogene and genes present onthe same chromosome constitutes a DR haplotype (FIG. 5). Each haplotypeis defined by the second DR molecule which characterizes it.

The role of the MHC II molecules in allergy was historically initiatedby the discovery of the association between the level of IgE against agiven allergen and certain alleles. These associations concern numerousallergens of low molecular mass such as Amb a5, Lol p3 and Amb a6 (31)which are allergens for which the relative risks are the greatest. Theseassociations are not systematic or correspond to very low relativerisks.

In the case of bee venom, it has been shown that the HLA-DR7 allele ismore frequent in allergic patients than in the control population (32)whereas the HLA-DR4 allele is by contrast underrepresented in thoseallergic to bee venom (33).

The control of the IgE (and IgG) response by the class II molecules iseven clearer in mice (34). The H-2^(d) and H-2^(k) mice are indeed goodresponders whereas the H-₂ ^(b) mice respond little or not at all tothis allergen.

The subject of the present invention is peptides capable ofdesensitizing a subject allergic to bee venom, characterized in thatthey are selected from the group consisting of:

fragment (1) corresponding to positions P85-97 of the major bee venomallergen,

fragment (2) corresponding to positions P81-93 of the major bee venomallergen,

fragment (3) corresponding to positions P94-106 of the major bee venomallergen,

fragment (4) corresponding to positions P76-88 of the major bee venomallergen,

fragment (5) corresponding to positions P77-94 of the major bee venomallergen,

fragment (6) corresponding to positions P122-134 of the major bee venomallergen, and

the mutated fragments of said fragments (1) to (6) which exhibit an MHCclass II molecule binding activity identical to or higher than those ofsaid fragments (1) to (6).

Fragments (1) and (2) form group I; fragment (3) forms group II;fragments (4) and (5) form group III and fragment (6) forms group IV.

Such peptides comprise T epitopes or determinants which interact withone or more HLA-DR molecules derived from the DRB1 genes or other DRBs(DRB3, DRB4 and DRB5).

The present invention also includes the peptides as defined above,polymerized.

The site for binding of the peptides to the class II molecules issituated between the α helices of the α1 and α2 domains and forms agroove which is open at both ends. This opening allows the binding ofpeptides with varying sizes, in general from 13 to 25 amino acids. Theanchoring of the peptides to the MHC II molecules occurs by means ofhydrogen bonds between the backbone of the peptide and the amino acidsof the groove and by means of residues accommodated by pockets forspecificity. Five pockets, called P1, P4, P6, P7 and P9, correspond tothe amino acid of the peptide which it accommodates, the first positionbeing that which is in the first pocket, receive amino acids of thepeptide and are composed of conserved or polymorphic residues. Thepolymorphic residues are responsible for various specificities betweenMHC II molecules. Since the binding site is open, two peptides bindingone MHC II molecule may do so according to different modes, that is tosay using different anchoring residues in their sequence.

Peptides P81-93 and P85-97, mutated in at least one residue, correspondto one of the pockets P1, P4, P6, P7 and P9 are in particular includedin the invention.

In order to be able to introduce residues which preserve or increase thebinding activity, the modes of interaction of the peptides P81-93 andP85-97 with respect to the HLA-DR molecules capable of binding them werestudied. The approach chosen was to introduce into each position analanine so as to evaluate the role of the side chain in the interactionor a lysine which is a basic and bulky amino acid. If necessary, acombination of mutations was introduced. By way of example, a reductionin activity caused by substitutions of phenylalanine 88 by a lysine oran alanine is observed in FIG. 6. Other reductions in activity areobserved a positions situated at 3, 5 and 8 amino acids fromphenylalanine 88. This activity profile corresponds to a mode of bindingwhere positions F88, I91 T93 and Y96 are accommodated by the pockets P1,P4, P6 and P9, respectively, of the molecules HLA-DRB3*0101,HLA-DRB5*0101, HLA-DRB1*1301 and HLA-DRB1*0701. This mode of associationwas confirmed by molecular modeling for the complexes: P85-97/DRB3*0101,P85-97/DRB5*0101 and P81-93/DRB4*0101. All the results are given in FIG.7. It is observed that on sequence 81-97, there are at least six modesof binding to the HLA-DR molecules. It is also observed that mode I iscommon to eight molecules whereas modes V and VI are specific to asingle molecule.

The fact that it is possible to know exactly how the peptide ispositioned on each HLA-DR molecule makes it possible to propose sequencemodifications:

at positions P1, P4, P6, P7 and P9, it is possible to introducemodifications which are compatible with the known specificities of theHLA molecules (61). It is for example probable that tyrosine 87 can bechanged by a phenylalanine without any loss of activity. On the otherhand, the substitution of phenylalanine 88 by a tyrosine should notcause a loss of activity for, for example, the molecules DRB1*0101 andDRB1*0401 but should reduce the activity of the peptide for moleculessuch as DRB1*0301 or DRB3*0101.

it is also possible to increase the affinity of the peptide bymodifying, in an optimum manner, residues P1, P4, P6, P7 and P9. This iswhat was done for residues 89 and 84. Residue 89 can be advantageouslymodified to leucine or to threonine (Table IX). In particular, thesubstitution by leucine increases the affinity of the peptide 85-97 by afactor of 10 and 12 for the molecules DRB3*0101 and DRB1*0301,respectively. In the case of DRB1*0301, this increase is explained bythe fact that asparagine N89 at position P1 is not optimum for thismolecule, unlike leucine (61). Likewise, the substitution of glycine 84by an alanine increases the affinity by a factor of 5 for DRB1*1501.This increase is in agreement with the positioning of glycine 84 in P1and ought to be even higher if a hydrophobic residue such as leucine orisoleucine is introduced. On the other hand, the substitution ofaspartic acid D92 systematically reduces the affinity for DRB1*0301.This reduction in activity is in accordance with the anchoring role ofaspartic acid in P4 for this molecule.

at positions P-2, P-1, P2, P3, P5, P8, P10 and P11, modifications may beintroduced without fear of causing major losses in affinity.

TABLE IX Relative activities of the modified peptides relative to thepeptide P85-97A Peptides N89L N89T D92N D92L D92T DRB1*0101 2 1 2 1 2DRB1*0301 12 7 −57 −29 −12 DRB1*0401 4 2 3 −2 4 DRB1*0701 4 3 3 12 9DRB1*1101 8 2 8 7 13 DRB1*1301 1 1 1 2 1 DRB3*0101 10 2 1 2 2 DRB5*01011 −1 2 2 2

The peptides N89L, N89T, D92N, D92L, D92T are analogs of the peptideP85-97A. The first letter corresponds to the initial amino acid, thenumber to its position in the Api m1 sequence and the second letter tothe modification introduced. The data correspond to the ratios betweenthe IC₅₀ values for the analogs and that for the reference peptideP85-97A. A—sign means a loss of activity and the absence of a sign meansan increase in activity.

The peptide P85-97A possesses an alanine at position 95 as a replacementfor cysteine. This substitution does not cause any modification inactivity regardless of the HLA-DR molecule.

In accordance with the invention, at least one of positions P1, P4, P6and P9 of the fragments (1), with reference to FIG. 7 is mutated.

According to an advantageous feature of this embodiment, said fragments(1) comprise one of the following mutated amino acids: N89L, N89T, C95A,G84L, G84I.

Also in accordance with the invention, at least one of the positionsP-2, P-1, P2, P3, P5, P8, P10 and P11 of the fragments (1), withreference to FIG. 7, is mutated.

The subject of the present invention is also desensitizing compositionsfor bee venom allergies, characterized in that they comprise:

at least one peptide selected from group A consisting of:

the peptides of group I, as defined above, and

the peptides consisting of fragments of at least 13 amino acids whichare included in or comprise the fragment corresponding to positionsP81-97 of the major bee venom allergen (API m1) and which bind at leastto the HLA-DR molecules encoded by the HLA alleles DRB1*0101, DRB1*0301,DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301 and DRB1*1501 (molecules DR1,DR3, DR4, DR7, DR11, DR13 and DR2), with a binding activity <1000 nM,and

at least one pharmaceutically acceptable vehicle.

Such compositions which are particularly suitable for the patient may beadvantageously used preventively or curatively.

According to an advantageous embodiment of said compositions, theypreferably consist of a mixture of peptides comprising:

at least one group A peptide as defined above and at least one otherpeptide selected from the following groups:

the peptides selected from group B consisting of:

the peptides of group II, as defined above, and

the peptides consisting of fragments of at least 13 amino acids whichare included in or comprise the fragment corresponding to positions94-106 of the major bee venom allergen (API m1) and which bind at leastto the HLA-DR molecules expressed by the alleles DRB1*0101, DRB1*0401and DRB1*1101 (molecules DR1, DR4 and DR11), with a binding activity<1000 nM,

the peptides selected from group C consisting of:

the peptides of group III, as defined above, and

the peptides consisting of fragments of at least 13 amino acids whichare included in or comprise the fragment corresponding to positionsP76-94 of the major bee venom allergen and which bind at least to theHLA-DR molecules expressed by the alleles DRB1*0701, DRB1*1101 andDRB1*1501 (molecules DR7, DR11 and DR2), with a binding activity <1000nM, and

the peptides selected from group D consisting of:

the peptides of group IV, as defined above, and

the peptides consisting of fragments of at least 13 amino acids whichare included in or comprise the fragment corresponding to positionsP122-134 of the major bee venom allergen and which bind at least to theHLA-DR molecules expressed by the alleles DRB1*1101, DRB1*1301 andDRB1*1501 (molecules DR11, DR13 and DR2), with a binding activity <1000nM.

According to another embodiment of said compositions, they comprise, inaddition, optionally the peptide corresponding to positions P18-30and/or the peptide corresponding to positions P45-62 and/or the peptidecorresponding to positions P57-74 and/or the peptide corresponding topositions P65-82 and/or the peptide corresponding to positions P111-123,of the major bee venom allergen, in accordance with Tables IIIa and IIIbbelow and/or the peptides including the abovementioned peptides.

TABLE IIIa Peptides representative of the various zones of interactionbetween the major bee venom allergen and the HLA-DR molecules (1st gene)encoded by the DRB1 gene 101 401 1101 701 301 1301 1501 P18-30 P45-62P57-74 P65-82 P76-88 P77-94 P77-94 P81-93 P85- P85- P85-97 P85-97 P85-97P85-97  97 97 P94- P94- P94- 106 106 106 P111- 123 P122- P122-134 P122-134  134

TABLE IIIb Peptides representative of the various zones of interactionbetween the major bee venom allergen and the HLA-DR molecules (2nd gene)in which the β chain is not encoded by the DRB1 gene HLA-DR alleles (2ndgene) Cumulative B5*0101 B4*0101 B3*0101 frequency^(a) 21-38 7.9 45-627.9 53-70  9.2 77-94 77-94  17.1 81-93 28.4 85-97 85-97^(a) 17.1  89-10128.4  94-106 7.9 111-123 111-123 111-123 45.5 122-134 122-134 122-13445.5

These peptides are representative of the zones of interaction with the2nd HLA-DR molecules. They are part of the most active peptides for agiven zone and are as short as possible.

^(a): the cumulative frequencies between the alleles of various genesonly make sense because the 2nd molecules behave, within the Caucasianpopulation, like an identical allelic series.

Advantageously, in said compositions:

the group I peptides may be advantageously concatenated to form a singlepeptide (P81-97) and/or

the group III peptides may be advantageously concatenated to form asingle peptide (P76-94) corresponding to positions P81-97 of the majorbee venom allergen and/or

the group II and group III peptides may be advantageously concatenatedto form a single peptide (P76-106) corresponding to positions P81-97 ofthe major bee venom allergen.

In general, the compositions according to the invention comprise atleast one peptide including at least one of those described in Table IIIand which are suitable for the patient to be desensitized.

These desensitizing compositions are defined from the activities forbinding to the HLA-DR molecules of the peptides which they comprise,from the frequency of alleles toward which they are active and from thecomplementarity of the zones for interaction or epitopes which saidpeptides carry.

For a patient for whom the HLA-DR molecules which they are carrying arenot known, a composition according to the invention will be preferablyused which comprises at least one group A peptide.

There may be added thereto, advantageously and in order to increase thenumber of epitopes: a group B peptide; a group C peptide; a peptidecorresponding to the concatenation of a group B peptide and of a group Cpeptide and/or a group D peptide.

For the patients for whom the HLA molecules are known, there will beused either the peptide defined above, or a combination of peptidesincluding at least those described in Tables IIIa and IIIb, whichcorrespond to the alleles which the patient possesses.

The peptides included in said compositions were advantageously selectedusing an HLA-DR/peptide binding test comprising (i) incubating thepurified HLA-DR molecules selected from those relating to more than 5%of a given population and in particular the HLA molecules DR1, DR3, DR4,DR7, DR11, DR13 and DR2, simultaneously with various concentrations offragments of 13 to 18 amino acids which overlap and which completelycover the API m1 sequence and with a reagent R1 consisting of a peptidefragment combined with a nonradioactive marker, such as biotin and whosesequence is different from said peptides and is chosen such that itexhibits affinity toward the chosen HLA-DR molecule, such that it can beused at a concentration <200 nM, (ii) transferring the complexesobtained on an ELISA-type plate, previously sensitized with an antibodyspecific for all the HLA-DR molecules, (iii) revealing the HLA-DRmolecules/R1 reagent complexes, attached to the bottom of the plate bymeans of suitable conjugates, such as streptavidin-phosphatase and afluorescent substrate, (iv) selecting the peptides comprising differentepitopes, that is to say the most representative of the various zones ofinteraction between the major bee venom allergen and the HLA-DRmolecules and (v) choosing the most suitable peptides as a function ofthe frequency of the alleles toward which they exhibit a bindingactivity <1000 nM, corresponding to the concentration of this peptidewhich inhibits 50% of the binding of the reagent R1 (IC₅₀).

These tests make it possible, unambiguously, to combine with each alleleof the 1st gene or of the 2nd gene, the sequences of the fragmentscapable of binding thereto or on the contrary which do not bind thereto.

This approach makes it possible to define desensitizing compositionsincluding peptides which bind to the largest number of different HLA-DRmolecules and which can thus be advantageously desensitized for themajority of patients, even if their HLA molecules are not known.

This approach has, in addition, the advantage of allowing the selectionof peptides which are significantly more specific with respect to mostof the allergic subjects than the approaches seeking to select peptideson the basis of their capacity to stimulate T lymphocytes of allergicsubjects.

Thus, the inventors have found that only some peptides have a bindingactivity with respect to several of the most frequent alleles in theCaucasian population in accordance with Tables IIIa and IIIb.

The subject of the present invention is also desensitizing compositionsfor bee venom allergies, characterized in that they comprise at leastone modified bee venom phospholipase A2, in which the zones comprisingthe peptides as defined above are conserved and the zones outside theabovementioned zones are modified, such that they no longer exhibitreactivity toward the IgEs and at least one pharmaceutically acceptablevehicle.

Said zones are in particular modified by point mutation or deletion. Beevenom PLA2 is indeed capable of receiving numerous mutations anddeletions (53, 54). It is therefore possible to obtain mutants no longerexhibiting reactivity toward IgEs, such as those obtained for Bet VI(55). For example, the bee PLA2 mutant in which the lysine at position25 has been substituted is a lot less antigenic for apiarist sera thanthe native molecule (55).

The subject of the present invention is also the use of a modified beevenom phospholipase A2 in which the zones comprising the peptides asdefined above are conserved and the zones outside the abovementionedzones are modified, such that they no longer exhibit reactivity towardthe IgEs, for the preparation of a desensitizing composition for beevenom allergies.

The peptides which can be included in a desensitizing composition forbee venom allergies may be advantageously selected by a method whichcomprises:

(i) incubating the purified HLA-DR molecules selected from thoserelating either to less than 5% of a given population, that is to saythose consisting of HLA-DRs other than the HLA molecules DR1, DR3, DR4,DR7, DR11, DR13 and DR2, or HLA-DR molecules from a given patient,simultaneously with various concentrations of fragments of 13 to 18amino acids which overlap and which completely cover the API m1 sequenceand with a reagent R1 consisting of a peptide fragment combined with anonradioactive marker such as biotin and whose sequence is differentfrom the peptides, as defined above (groups A to D) and is chosen sothat it exhibits affinity toward the chosen HLA-DR molecule, such thatit can be used at a concentration <200 nM, (ii) transferring thecomplexes obtained on a microtiter plate, previously sensitized with anantibody specific for all the HLA-DR molcules, (iii) revealing theHLA-DR molecules/R1 reagent complexes, attached to the bottom of theplate by means of suitable conjugates, such as streptavidin-phosphataseand a fluorescent substrate, (iv) selecting the peptides comprisingdifferent epitopes, that is to say the most representative of thevarious zones of interaction between the major bee venom allergen andthe HLA-DR molecules studied and (v) choosing the most suitable peptidesas a function of the frequency of the alleles toward which they exhibita binding activity <1000 nM, corresponding to the concentration of thispeptide which inhibits 50% of the binding of the reagent R1 (IC₅₀).

The incubation conditions are specific to each HLA-DR molecule(incubation time, pH, reagent R1, HLA-DR or peptide concentration).

The reagent R1 is selected from the group consisting of the followingsequences:

PKYVKQNTLKLAT (SEQ ID NO: 1), specific for the alleles DRB1*0101,DRB1*0401, DRB1*1101,

EAEQLRAYLDGTGVE (SEQ ID NO: 2), specific for the allele DRB1*1501,

AKTIAYDEEARGLE (SEQ ID NO: 3), specific for the allele DRB1*0301,

AAYAAAKAAALAA (SEQ ID NO: 4), specific for the allele DRB1*0701,

TERVRLVTRHIYNREE (SEQ ID NO: 5), specific for the allele DRB1*1301,

ESWGAVWRIDTPDKLTGPFT (SEQ ID NO: 6), specific for the alleles DRB1*1301,DRB3*0101, and

AGDLLAIETDKATI (SEQ ID NO: 7), specific for the alleles DRB1*0701 andDRB4*0101.

Other reagents R1 may be used, in particular those described inSouthwood et al. (52).

To study the HLA-DR molecules (2nd gene), this requires appropriatepairs of biotinylated peptides which should bind the preparation at lowconcentration and be selective for one of the two molecules. Moreprecisely, the binding of the biotinylated peptides should beeffectively inhibited by their nonbiotinylated homolog, but not greatlydisrupted by the nonbiotinylated form of the other peptide (Table X). Ithas been possible to find such peptides for each of the moleculesDRB3*0101, DRB4*0101 or DRB5*0101.

Using these tests, the peptides of Api m1 which bind these moleculeswere defined. The same set of peptides as above was used: peptides of 18amino acids which cover the sequence of the allergen and peptides of 13residues which exhaustively cover particular zones. The results obtainedare illustrated in Table X below.

The DRB5*0101 allele interacts with seven different regions of Api m1.Only four and five regions of Api m1 can bind the alleles DRB4*0101 andDRB3*0101, respectively. They are mainly situated in the central andC-terminal part of the allergen. Peptides P111-123 and 122-134 indeedbinds the three second molecules. It is also advantageous to note thatpeptides common to the 1st and 2nd DR molecules, in particular thepeptides P81-93 and P85-97, exist.

TABLE X Sensitivity and selectivity of the biotinylated peptide tracers1st DRs 2nd DRs Peptides Allele IC₅₀ (nM) Allele IC₅₀ (nM) B1 21-36B1*1301 275 B3*0101 35000 LOL 191-210 B1*1301 >100000 B3*0101 5 YKLB1*0701 35 B4*0101 950 E2/E168 B1*0701 5000 B4*0101 2 A3 152-166 B1*150133 B5*0101 85000 HA 306-318 B1*1501 2500 B5*0101 6.5

The selectivity of each peptide is evaluated by the IC₅₀ of thenonbiotinylated peptide tested on each of the molecules. For each pair,the first peptide is that which is selective for the 1st DR molecule andthe second for the 2nd DR molecule.

The sequences of the peptides are as follows: B1 21-36

(TERVRLVTRHIYNREE) (SEQ ID NO: 5) (62), LOL 191-210

(ESWGAVWRIDTPDKLTGPFT) (SEQ ID NO: 6) (63), YKL

(AAYAAAKAAALAA) (SEQ ID NO: 4) (64), HA 306-318

(PKYVKQNTLKLAT) (SEQ ID NO: 1) (65), A3 152-166

(EAEQLRAYLDGTGVE) (SEQ ID NO: 2) (65), E2/E168

(AGDLLAIETDKATI) (SEQ ID NO: 7).

Surprisingly, the method of selection according to the invention issuitable for any HLA-DR molecule.

The subject of the invention is also a kit for selecting peptidescapable of desensitizing a subject allergic to bee venom, characterizedin that it comprises various concentrations of fragments of 13 to 18amino acids which overlap and which completely cover the API m1sequence, a set of reagents R1 each consisting of a peptide fragmentcombined with a non-radioactive marker, such as biotin and whosesequence is different from the peptides as defined above (groups A to D)and is chosen such that it exhibits affinity toward the chosen HLA-DRmolecule such that it can be used at a concentration <200 nM and anantibody specific for all the HLA-DR molecules.

In addition to the preceding features, the invention further comprisesother features which will emerge from the description which follows,which refers to exemplary embodiments of the method which is the subjectof the present invention, as well as to the accompanying drawings inwhich:

FIG. 1 represents the protein sequence of bee venom phospholipase A2(API m1) (SEQ ID NO: 8), deduced from that of the complementary DNA,

FIG. 2 illustrates the activity for binding to the HLA-DR molecules ofthe peptides of thirteen amino acids which cover sequence 15-37 of themajor bee venom allergen. The results are expressed in the 1/IC₅₀ form.The unit is M⁻¹;

FIG. 3 illustrates the activity for binding to the HLA-DR molecules ofthe peptides of thirteen amino acids which cover sequence 107-134 of themajor bee venom allergen. The results are expressed in the 1/IC₅₀ form.The unit is M⁻¹;

FIG. 4 illustrates the frequencies of the determinants of API m1 in theCaucasian population. This representation is based on the frequencies ofthe alleles studied in the French population. In other Caucasianpopulations, substantially the same profile is found;

FIG. 5 illustrates the structure of the genes for the MHC of the HLA-DRclass per haplotype; in this figure, the genes are schematicallyrepresented by black boxes and the pseudogenes by hatched blocks. Thename of the genes is given above the block and that of the principalalleles present in the haplotype is given under the block;

FIG. 6 illustrates the location of the residues for anchoring thepeptide P85-97 on the HLA-DR molecules. Each position was substituted byan alanine or a lysine. The results are given as relative activity withrespect to the peptide P85-97A (0.01 means a relative loss by a factorof 100);

FIG. 7 illustrates the modes of binding of the sequence 81-97 to theHLA-DR molecules. The blocks schematically represent the site of bindingof the HLA-DR molecules which characterize the pockets P1, P4, P6, P7and P9. A color and a number in Roman numeral was attributed to eachmode so as to facilitate their visualization; and

FIG. 8 illustrates the T lymphocyte response of patients allergic to beevenom, to the peptides which cover the Api m1 sequence; the PBMCs of theallergic patients were cultured for 7 days in the presence of Api m1. AtD=7 and D=10, IL2 (50 U/ml) was added. At D=14, the cells were harvestedand then incubated in the presence of various peptides. Theproliferation of the cells was estimated by incorporating tritiatedthymidine. Composition of the pools: pool 1: P1-18, P5-22, P9-26, pool2: P13-30, P17-34, P21-38, pool 3: P25-42, P29-46, P33-50, P37-54, pool4: P41-58, P45-62, P49-66, pool 5: P53-70, P57-74, pool 6: P61-78,P65-82, P69-86, P73-90, pool 7: P77-94, P81-98, P85-102, P89-106,P93-110, pool 8: P97-114, P101-118, pool 9: P105-122, P109-126,P113-130, P117-134.

It should be understood, however, that these examples are given by wayof illustration of the subject of the invention and do not in any mannerconstitute a limitation thereto.

EXAMPLE 1 Principle of the Binding Tests

Synthesis of the Peptides

The peptides which cover the sequence of the major bee venom allergen(FIG. 1) were chosen from the sequence deduced from that of thecorresponding complementary DNA (36). All the peptides were synthesizedaccording to the Fmoc strategy in solid phase parallel synthesis,purified by HPLC and checked by mass spectrometry (ES-MS).

Purification of the HLA-DR Molecules

The HLA-DR molecules are purified from various homozygous EBV lines (52)by immunoaffinity. It is possible in particular to use the methoddescribed in Southwood et al. (52). Their origin and the various alleleswhich characterize them are described in Table IV.

TABLE IV Other DRB Lines Specificities DRB1 alleles alleles LG2 (52)HLA-DR1 DRB1*0101 — HOM2 SCHU HLA-DR2 DRB1*1501 DRB5*0101 MAT (52)HLA-DR3 DRB1*0301 DRB3*0101 STEILIN BOLETH HLA-DR4 DRB1*0401 DRB4*0101PREISS (52) PITOUT (52) HLA-DR7 DRB1*0701 DRB4*0101 SWEIG (52)  HLA-DR11DRB1*1101 DRB3*0202 HHKB (46)  HLA-DR13 DRB1*1301 DRB3*0101

The hybridomas secreting a monomorphic antibody specific for the HLA-DRmolecules is in particular that described in Southwood et al. (52) orthat described in Posch et al. (42). The antibodies are purified fromculture supernatants on Protein A-Sepharose columns. These antibodiesare coupled onto Sepharose 4B or Protein A-Sepharose columns for thepurification of the HLA-DR molecules.

Tests for HLA-DR/Peptide Binding

The tests for binding of the peptides to the HLA-DR molecules arecompetition tests with immunoenzymatic visualization, initiallydeveloped by Hill on the HLA-DR molecule (37). They are carried out in96-well plates, which makes it possible to study numerous samples in thesame experiment. Briefly, the purified HLA-DR molecules are incubatedwith a biotinylated peptide which serves as tracer and variousconcentrations of the peptide to be tested.

After incubating for 24 to 72 hours, the samples are neutralized, andthen 100 μl of each sample are transferred onto an ELISA platepreviously sensitized by the monomorphic antibody specific for theHLA-DR molecules. The HLA-DR molecules/biotinylated peptide complexesattached at the bottom of the plate via the monomorphic antibodyspecific for the HLA-DR molecules are visualized by means of thestreptavidin-phosphatase conjugate and a fluorescent substrate. Theactivity of each peptide is characterized by the concentration of thispeptide which inhibits the binding of the biotinylated peptide by 50%(IC₅₀).

Choice and Optimization of the Binding Tests

Choice of the alleles (1st gene)

The alleles studied are all the alleles in the French population whosefrequency exceeds 5% of the population.

They are the alleles DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701,DRB1*1101, DRB1*1301 and DRB1*1501 (Table I). They represent, on theirown, 53 to 82% of the alleles in the Caucasian populations and form partof the various specificities of the HLA-DR series.

Choice of the alleles (2nd gene)

The alleles studied are the alleles most frequently encountered. Theyare the alleles HLA-DRB3*0101, HLA-DRB4*0101 and HLA-DRB5*0101 (seeTable IIIb).

Specificity of the tests

The choice of the biotinylated peptides is the key element in thespecificity of the test. Most of the cells used possess two differentHLA-DR molecules which are both purified by a monomorphic antibodyspecific for the HLA-DR molecules and both are recognized by the sameantibody. In order to unambiguously study the binding of a peptide tothe DRB1 allele, it is necessary to ensure that the biotinylated peptidebinds this allele and does not bind the product of the other gene.

For this purpose, a number of peptides, which are described in Table V,were used.

TABLE V 2nd gene 1st gene (alleles (alleles DRB3, DRB4, Refer- ReagentR1 Sequences DRB1) DRB5) ences HA 306-318 SEQ ID NO:1 DRB1*0101DRB5*0101 37-39 (=HA 307- DRB1*0401 37, 40, 319) 41, 42 DRB1*1101 38,39, 42 DRB1*1501 A3 152-166 SEQ ID NO:2 DRB1*1501 DRB5*0101 43 MT 2-16SEQ ID NO:3 DRB1*0301 44 YKL SEQ ID NO:4 DRB1*0701 DRB4*0101 41 B1 21-36SEQ ID NO:5 DRB1*1301 DRB3*0101 46 LOL 191- SEQ ID NO:6 DRB1*1301DRB3*0101 63 210 E2/E168 SEQ ID NO:7 DRB1*0701 DRB4*0101

For HLA-DRB1*0101, DRB1*0401 and DRB1*1101, the peptide ha 306-318 whichother authors had previously used in tests for binding to these alleles(37, 41), was used.

Likewise, for DRB1*0301 and DRB1*1501, the tracers used (reagent R1)derive from tracers which have already been described (44), (45).

For DRB1*0701, the peptide YKL has been described as being a very goodligand (41) whereas the peptide B1 21-36 is a natural ligand forDRB1*1301 (46). Neither of them has already been used as tracer.

Test conditions and sensitivity

For each HLA-DRB1 molecule, the concentration of MHC II molecules, theconcentration of the biotinylated peptide, the incubation pH and theincubation time were optimized. For the molecules HLA-DRB1*101,HLA-DRB1*0401 and HLA-DRB1*0701, the conditions for the ELISA tests aresimilar to those already described by other authors (37, 41). For theother HLA-DRB1 molecules which were studied, there are no ELISA testsdescribed in the literature. The details of the conditions used aredescribed in Table VI.

TABLE VI Protein Tracer concen- concen- Incubation tration trationOptimum time Alleles (μg/ml) Tracers (nM) pH (h) DRB1*0101 0.6 HA306-318 10 6 24 DRB1*0301 2.3 MT 2-016 50 4.5 72 DRB1*0401 1.6 HA306-318 30 6 24 DRB1*0701 0.4 YKL 10 5 24 DRB1*1101 1.3 HA 306-318 20 524 DRB1*1301 0.7 B1 21-36 200 4.5 72 DRB1*1501 0.5 A3 152-166 10 4.5 24

The sensitivity of each test is reflected by the IC₅₀ values observedwith the nonbiotinylated peptides which correspond to the tracers (TableVII and Table X).

TABLE VII

Activity for binding to the HLA-DR molecules of the peptides of eighteenamino acids which cover the sequence of the major bee venom allergen(SEQ ID NO: 8).

-: activity ≧100000 nM

The results are expressed in the form of the IC₅₀. The unit is nM. TheIC₅₀ values of the best peptides (IC₅₀ less than 1000 nM) have beenboxed.

They vary from 14 to 44 nM for the alleles 101, 401, 1101, 701 and 1501which are very satisfactory values. They are only 100 to 320 nM for thealleles 301 and 1301, respectively, and indicate a sensitivity ofaverage quality.

Mapping of the Determinants of API m1 Restricted to the HLA-DR MoleculesStudied

Localization of the determinants of API m1: by means of peptides of 18residues

The peptides which are naturally present on the HLA-DR molecules havesizes which vary between 13 and 25 amino acids approximately, the sizemost frequently encountered being 15 amino acids. In order to optimizethe search for the peptides of API m1 which are capable of binding tothe HLA-DR molecules, we synthesized thirty peptides of 18 amino acidswhich overlap by 14 amino acids and which therefore contain all thepeptides of 15 possible residues of the API m1 sequence. The bindingcapacities of each of these peptides were tested on the 7 alleles whichwe selected and are expressed in the IC₅₀ form (Table VII).

Alleles 101, 401 and 1101 have a very similar activity profile and inparticular accept the 4 peptides (P81-98, P85-102, P89-106 and P93-110)as ligands. However, they-exhibit differences. The peptide P105-122indeed binds the allele 401, whereas the peptides P77-94 and P117-134bind the allele 1101. The allele 701 possesses 7 active peptides(P13-30, P17-34, P21-38, P45-62, P77-94, P81-98 and P85-102) whereas 4peptides (P81-98, P85-102, P53-70 and P57-74) are active toward allele301. The peptides P81-98 and P85-102 bind allele 1301 with an affinitycomparable to that of the peptide P117-134. Finally, two active regionsexist for allele 1501 and comprise, on the one hand, peptides P65-82,P69-86, P73-90, P77-94, P81-98 and P85-102 and, on the other hand,peptides P113-130 and P117-134.

Clearly, each allele possesses a profile for binding of the peptides ofAPI m1 which is specific to it. Some peptides bind to only one allele(for example P105-122 to allele 401, P69-86 and P73-90 to allele 1301).Others, by contrast, bind to several alleles. That is the case inparticular for peptide P81-98 which significantly binds the sevenalleles studied and for peptide P85-102 which significantly binds sixalleles. In many cases, a peptide is common to two or several alleles.These common peptides define mainly three distinct zones of the API m1sequence: i) an N-terminal part which the peptides P13-30, P17-34,P21-38 form, ii) a central part consisting of the peptides P77-94 toP93-110 and iii) a C-terminal part which includes the peptides P113-122to P117-134. It is also observed that thirteen peptides out of thethirty tested have binding activities >1000 nM, regardless of the MHC IImolecule studied. They are therefore inactive or not very active.

Table VIII below illustrates the activity for binding to the HLA-DRmolecules of the peptides of 13 amino acids which cover the zone 73-108of API m1.

TABLE VIII

Activity for binding to the HLA-DR molecules of the peptides of 13 aminoacids which cover the zone 73-108 of the major bee venom allergen (SEQID NO: 8).

-: activity ≧100000 nM

The results are expressed in the form of the IC₅₀. The unit is nM. TheIC₅₀ values of the best peptides (IC₅₀ less than 1000 nM) have beenboxed.

Precise location of the determinants of API m1

In order to better define the zones of contact between the activepeptides and the MHC II molecules, all the peptides of thirteen residueswhich cover the active zones were synthesized. The size of the thirteenamino acids was chosen as a compromise. It corresponds to the minimumsize of the peptides which are naturally present on the MHC II moleculesand should be sufficiently small to discriminate between two contactsurfaces contained in a single peptide of eighteen amino acids.

11 peptides which exhaustively cover the N-terminal part 15 to 37 weretested on the alleles 101, 401, 701, 1101 and 1301 (FIG. 2). For theallele 701, the peptides P18-30 to P22-34 exhibit a significant bindingactivity which, in the case of the peptide P18-30, is equivalent to thatof the peptide of eighteen amino acids P21-38.

The alleles 401, 101 and 1101 substantially bind the same peptides asthe allele 701. However, the binding activities observed are lower,which is in agreement with those obtained for the peptides of eighteenamino acids.

The central part was studied on the seven alleles by means oftwenty-four peptides of thirteen residues. For the allele 101, thepeptides P85-97, on the one hand, and P91-103 to P95-107, on the otherhand, define two distinct zones of interaction. These two zones are alsofound for the alleles 401 and 1101 but with small variations. The firstcomprises, in addition to the peptide P85-97, the peptides P82-94 andP83-95 for the allele 401 and the peptide P83-95 for the allele 1101.The second zone of contact is strictly identical between the allele 401and 101 whereas it is reduced to a single peptide (P94-106) for theallele 1101. The allele 701 is characterized by three peptides havinggood binding activity (P85-97, P86-98 and P87-99) which corresponds tothat of the peptides of eighteen amino acids P81-98 and P85-102. Thegood activity for binding of the peptide P77-94 to the allele 701previously described is not observed for any of the peptides of thirteenresidues which cover this zone, the peptides P76-88 to P81-93 having asubstantially lower activity.

The peptide P85-97 possesses, for the alleles 301 and 1301, a bindingactivity similar to that of the peptides of 18 amino acids P81-98 andP85-102, containing it. The allele 1301 also accepts the peptide P86-105as ligand. For the allele 1501, seven peptides, including six which areconsecutive (P73-85 to P78-90) and one which is isolated (P81-93)reflect the activity of the three peptides of eighteen amino acids(P73-90, P77-94 and P81-98)

Finally, the C-terminal part of API m1 was studied for the alleles 401,1501, 1101 and 1301 by means of sixteen peptides of thirteen residues.The allele 401 is characterized by six active peptides (P109-121 toP114-126) whereas seven other peptides (P116-128 to P122-14) bind with ahigh affinity to 1501. The peptides P121-133 and 122-134 are also veryactive for the alleles 1101 and 1301.

These results describe the precise location of the binding determinantsof the major bee venom allergen for the seven alleles chosen and clearlyshow the differences and the similarities between the alleles for thevarious peptides of API m1. In order to better account for the activityand the location of these determinants, a representative peptide, whichis as short as possible and whose binding activity reflects that of thedeterminant (Table III) was chosen for each of them. It will be notedmost particularly that the peptide P85-97 is representative of adeterminant for the alleles 101, 401, 1101, 701, 301 and 1301 and thatother determinants are advantageously situated near this peptide(P76-88, P77-94, P81-93, P94-106).

Frequencies of the determinants of API m1 in the Caucasian population

To evaluate the impact of the most active peptides (activity less than1000 nM) within the Caucasian population, the cumulative frequency ofthe alleles which they are capable of binding (FIG. 4) was calculatedfor each. Applied to all the peptides of 18 amino acids which completelycover the sequence of the major allergen, this representation makes itpossible to weight the activity of the peptides tested. It is clearlyobserved that the central zone ranging from the peptide P77-94 to thepeptide P93-110 is that which has the greatest impact in the population.The peptide P81-98 binding with a good affinity to all the HLA-DRsstudied, covers on its own 63% of the population and combines the impactof the determinants carried by the peptides P85-97 and P81-93. Theaddition of sequences at the N- and C-terminals makes it possible to addto this combination of determinants other zones of interaction such asP76-88, P77-94 and P93-106 which relate to nonnegligible percentages ofthe population. Finally, the impact of the peptides P117-134 or 122-134greater than 20% is observed at the C-terminal.

EXAMPLE 2 Peptides Selected/cell Proliferation Correlation

The peptides which bind to the HLA molecules are not necessarilystimulating for the T lymphocytes. They can indeed resembleself-peptides, such that no T lymphocyte will be capable of recognizingthem. On the other hand, it has previously been shown that all thepeptides which stimulate the T lymphocytes are part of the best peptideswhich bind to the MHC II molecules (60). The binding to the MHC IImolecules is therefore a necessary but insufficient condition forallowing a peptide to be recognized by the T lymphocytes. In order toverify that the peptides which were identified are effectively capableof stimulating T lymphocytes in allergic patients, their stimulatingcapacity was tested (FIG. 8). The cells used are peripheral blood cellsfrom people allergic to bee venom. These people came to the RothschildHospital to be desensitized and agreed to participate in this study (DGSNo. 980457). Given the small number of cells, the peptides were groupedinto different pools. The cells from one patient did not result in anyproliferation (not shown). Of the six patients for whom reactivity wasobserved, a high variability was observed in the intensity of theresponse to the peptides, in the nature and the number of activepeptides. However, it is observed that of the 6 patients, 5 respond topool 7 which comprises peptides which cover zone 77-111 and 3 respond topool 9 which covers zone 105-134.

The other peptides can respond strongly but in a manner specific to agiven patient (example: pool 3 with the patient HD). Consequently, theseresults confirm the benefit of the use of the sequence 76-106 fordesensitization and, to a lesser degree, of the C-terminal region:105-134.

REFERENCES

1. MULLER U. R. et al., Monogr. Allergy, 1993, 31, 131.

2. MULLER U. R. et al., Clin. Exp. Allergy, 1998, 28, 4.

3. MULLER U. et al., J. Allergy Clin. Immunol., 1992, 89, 529.

4. KING T. P. et al., Arch. Biochem. Biophys., 1976, 172, 661.

5. V. LIEBERS et al., Clin. Exp. Allergy, 1996, 26, 494.

6. CHRETIEN I. et al., Eur. J. Immunol., 1990, 20, 243.

7. HUSSAIN R. et al., J. Immunol., 1992, 148, 2731.

8. ROCKLIN R. E. et al., N. Engl. J. Med., 1980, 302, 1213.

9. AKDIS C. A. et al., J. Clin. Invest., 1996, 98, 1676.

10. KAMMERER R. et al., J. Allergy Clin. Immunol., 1997, 100, 96.

11. JUTEL M. et al., J. Immunol., 1995, 154, 4187.

12. SECRIST H. et al., J. Exp. Med., 1993, 178, 2123.

13. MILICH D. R. et al., J. Immunol., 1989, 143, 3148.

14. BURSTEIN H. J. et al., J. Immunol., 1992, 148, 3687.

15. GERMAIN R. N. et al., Annu. Rev. Immunol., 1993, 11, 403.

16. BRINER T. J. et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 7608.

17. HOYNE G. F. et al., J. Exp. Med., 1993, 178, 1783.

18. BAUER L. et al., Clin. Exp. Immunol., 1997, 107, 536.

19. VRTALA S. et al., Int. Arch. Allergy Immunol., 1997, 113, 246.

20. PESCE A. J. et al., Int. Arch. Allergy Appl. Immunol., 1990, 92, 88.

21. LITWIN A. et al., Int. Arch. Allergy Appl. Immunol., 1988, 87, 361.

22. MULLER U., J. Allergy Clin. Immunol., 1998, 101, 747.

23. DHILLON M., J. Allergy Clin. Immunol., 1992, 90, 42.

24. CARBALLIDO J. M., J. Immunol., 1993, 150, 3582.

25. DUDLER T. et al., Eur. J. Immunol., 1995, 25, 538.

26. NORMAN P. S. et al., Am. J. Respir. Crit. Care Med., 1996, 154,1623.

27. TAKAI T. et al., Nat. Biotechnol., 1997, 15, 754.

28. SMITH A. M. et al., J. Allergy Clin. Immunol., 1998, 101, 423.

29. FERREIRA F. et al., Faseb J., 1998, 12, 231.

30. KAMMERER R. et al., Clin. Exp. Allergy, 1997, 27, 1016.

31. VAN NEERVEN R. J. et al., Immunol. Today, 1996, 17, 526.

32. FAUX J. A. et al., Clin. Exp. Allergy, 1997, 27, 578.

33. LYMPANY P. et al., J. Allergy Clin. Immunol., 1990, 86, 160.

34. LUCAS A. et al., Immunogenetics, 1986, 23, 417.

35. COLOMBANI J., 1993, John Libbey Eurotext, HLA: fonctionsimmunitaires et applications médicales [immune functions and medicalapplications].

36. KUCHLER K. et al., Eur. J. Biochem., 1989, 184, 249.

37. HILL C. M. et al., J. Immunol., 1994, 152, 2890.

38. ROCHE P. A. et al., J. Immunol., 1990, 144, 1849.

39. O'SULLIVAN D. et al., J. Immunol., 1990, 145, 1799.

40. SETTE A. et al., J. Immunol., 1993, 151, 3163.

41. MARSHALL K. W. et al., J. Immunol., 1994, 152, 4946.

42. POSCH P. E. et al., Eur. J. Immunol., 1996, 26, 1884.

43. VOGT A. B. et al., J. Immunol., 1994, 153, 1665.

44. GELUK A. et al., J. Immunol., 1992, 149, 2864.

45. SIDNEY J. et al., J. Immunol., 1992, 149, 2634.

46. DAVENPORT M. P. et al., Proc. Natl. Acad. Sci. USA, 1995, 92, 6567.

47. HAMMER J. et al., Adv. Immunol., 1997, 66, 67.

48. SHIPOLINI R. A. et al., Eur. J. Biochem., 1974, 48, 465.

49. MAILLERE B. et al., Mol. Immunol., 1995, 32, 1073.

50. MAILLERE B. et al., Mol. Immunol., 1995, 32, 1377.

51. COTTON J. et al., Int. Immunol., 1998, 10, 159.

52. SOUTHWOOD S. et al., J. Immunol., 1998, 160, 3363-3373.

53. NICOLAS J. P. et al., J. Biol. Chem., 1997, 272, 11, 7173-7181.

54. GHOMASHCHI F. et al., Biochem., 1998, 37, 6697-6710.

55. DUDLER T. et al., J. Immunol., 1994, 152, 5514-5522.

56. IRLE C. et al., J. Exp. Med., 1988, 167, 853.

57. FRIEDL-HAJEK R. et al., Clin. Exp. Allergy, 1999, 29, 478.

58. KIM J. et al., J. Immunol., 1997, 159, 335.

59. SHIMODA S. et al., J. Exp. Med., 1995, 181, 1835.

60. TEXIER C. et al., Int. Immunol., 1999, 11.

61. RAMMENSEE H. G. et al., Immunogenetics, 1995, 41, 178.

62. DAVENPORT M. P. et al., Proc. Natl. Acad. Sci. USA, 1995, 92, 6567.

63. SIDNEY J. C. et al., J. Immunol., 1992, 149, 2634.

64. MARSHALL K. W. et al., J. Immunol., 1994, 152, 4946.

65. VOGT A. B. et al., Immunol., 1994, 153, 1665.

As is evident from the above, the invention is not at all limited tothose of its embodiments, implementations and applications which havejust been described more explicitly; it encompasses, on the contrary,all the variants which may occur to the specialist in this field,without departing from the framework or the scope of the presentinvention.

8 1 13 PRT Artificial Sequence Fragment binding HLA-DR alleles 1 Pro LysTyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr 1 5 10 2 15 PRT ArtificialSequence Fragment binding HLA-DR alleles 2 Glu Ala Glu Gln Leu Arg AlaTyr Leu Asp Gly Thr Gly Val Glu 1 5 10 15 3 14 PRT Artificial SequenceFragment binding HLA-DR alleles 3 Ala Lys Thr Ile Ala Tyr Asp Glu GluAla Arg Gly Leu Glu 1 5 10 4 13 PRT Artificial Sequence Fragment bindingHLA-DR alleles 4 Ala Ala Tyr Ala Ala Ala Lys Ala Ala Ala Leu Ala Ala 1 510 5 16 PRT Artificial Sequence Fragment binding HLA-DR alleles 5 ThrGlu Arg Val Arg Leu Val Thr Arg His Ile Tyr Asn Arg Glu Glu 1 5 10 15 620 PRT Artificial Sequence Fragment binding HLA-DR alleles 6 Glu Ser TrpGly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr 1 5 10 15 Gly ProPhe Thr 20 7 14 PRT Artificial Sequence Fragment binding HLA-DR alleles7 Ala Gly Asp Leu Leu Ala Ile Glu Thr Asp Lys Ala Thr Ile 1 5 10 8 134PRT Apis mellifera 8 Ile Ile Tyr Pro Gly Thr Leu Trp Cys Gly His Gly AsnLys Ser Ser 1 5 10 15 Gly Pro Asn Glu Leu Gly Arg Phe Lys His Thr AspAla Cys Cys Arg 20 25 30 Thr His Asp Met Cys Pro Asp Val Met Ser Ala GlyGlu Ser Lys His 35 40 45 Gly Leu Thr Asn Thr Ala Ser His Thr Arg Leu SerCys Asp Cys Asp 50 55 60 Asp Lys Phe Tyr Asp Cys Leu Lys Asn Ser Ala AspThr Ile Ser Ser 65 70 75 80 Tyr Phe Val Gly Lys Met Tyr Phe Asn Leu IleAsp Thr Lys Cys Tyr 85 90 95 Lys Leu Glu His Pro Val Thr Gly Cys Gly GluArg Thr Glu Gly Arg 100 105 110 Cys Leu His Tyr Thr Val Asp Lys Ser LysPro Lys Val Tyr Gln Trp 115 120 125 Phe Asp Leu Arg Lys Tyr 130

What is claimed:
 1. A polypeptide molecule capable of desensitizing ahuman subject to bee venom, the polypeptide selected from the groupconsisting of: (a) a polypeptide molecule corresponding to amino acidpositions 85-97 of SEQ ID NO: 8; (b) a polypeptide moleculecorresponding to amino acid positions 81-93 of SEQ ID NO: 8; (c) apolypeptide molecule corresponding to amino acid positions 94-106 of SEQID NO: 8; (d) a polypeptide molecule corresponding to amino acidpositions 76-88 of SEQ ID NO: 8; (e) a polypeptide moleculecorresponding to amino acid positions 77-94 of SEQ ID NO: 8; (f) apolypeptide molecule corresponding to amino acid positions 122-134 ofSEQ ID NO: 8; and (g) a polypeptide molecule of (a)-(f) comprising atleast one amino acid substitution, wherein the substituted polypeptideexhibits binding activity to MHC class II molecules identical to orgreater than that of the polypeptides (a)-(f).
 2. A polypeptide moleculeof claim 1, wherein said at one amino acid sustitution occurs at aposition corresponding to amino acid position 83, 84 or 86 to 97 of SEQID NO:
 8. 3. A polypeptide molecule of claim 2, wherein the at least oneamino acid sustitution is selected from the group consisting of N89L,N89T, C95A, G84L and G84I.
 4. A polymer molecule comprising at least onepolypeptide of claim
 1. 5. A pharmaceutical composition fordesensitizing a human subject to bee venom, the composition comprising:at least one polypeptide molecule corresponding to amino acid positions85-97 of SEQ ID NO: 8 or to amino acid positions 81-93 of SEQ ID NO: 8;at least one polypeptide molecule of at least 13 amino acids andcorresponding to a consecutive amino acid sequence within the range ofamino acid positions 81-97 of SEQ ID NO: 8 and which binds to at leastone HLA-DR molecule encoded by the HLA alleles DRB1*0101, DRB1*0301,DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301 or DRB1*1501 with a bindingactivity <1000 nM; and at least one pharmaceutically acceptable vehicle.6. A pharmaceutical composition for desensitizing a human subject to beevenom comprising: (a) at least one polypeptide molecule corresponding toamino acid positions 85-97 of SEQ ID NO: 8 or to amino acid positions81-93 of SEQ ID NO: 8; (b) at least one polypeptide molecule of at least13 amino acids and corresponding to a consecutive amino acid sequencewithin the range of amino acid positions 81-97 of SEQ ID NO: 8 and whichbinds to at least one HLA-DR molecule encoded by the HLA allelesDRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301 orDRB1*1501 with a binding activity <1000 nM; (c) at least one polypeptidemolecule corresponding to amino acid positions 94-106 of SEQ ID NO: 8 ora polymer thereof; (d) at least one polypeptide molecule of at least 13amino acids and corresponding to a consecutive amino acid sequencewithin the range of amino acid positions 94-106 of SEQ ID NO: 8, themolecule binding to at least one HLA-DR molecule encoded by the allelesDRB1*0101, DRB1*0401 or DRB1*1101 with a binding activity <1000 nM; (e)at least one polypeptide molecule corresponding to amino acid positions76-88 or 77-94 of SEQ ID NO: 8 or a polymer thereof; (f) at least onepolypeptide molecule of at least 13 amino acids and corresponding to aconsecutive amino acid sequence within the range of amino acid positions76-94 of SEQ ID NO: 8, the molecule binding to at least one HLA-DRmolecule encoded by the alleles DRB1*0701, DRB1*1101 or DRB1*1501 with abinding activity <1000 nM; (g) at least one polypeptide moleculecorresponding to amino acid positions 122-134 of SEQ ID NO: 8 or apolymer thereof; (h) at least one polypeptide molecule of at least 13amino acids and corresponding to a consecutive amino acid sequencewithin the range of amino acid positions 122-134 of SEQ ID NO: 8, themolecule binding to at least one HLA-DR molecule encoded by the allelesDRB1*1101, DRB1*1301 or DRB1*1501 with a binding activity <1000 nM; and(i) at least one pharmaceutically acceptable vehicle, wherein anypolypeptide may contain at least one mutation such that the resultantmutant binds at least to one HLA-DR molecule encoded by the allelesDRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301 orDRB1*1501 with a binding activity <1000 nM.
 7. A pharmaceuticalcomposition of claim 6, further comprising at least one polypeptidemolecule selected from the group consisting of a polypeptide moleculecorresponding to amino acid positions 18-30 of SEQ ID NO: 8, apolypeptide molecule corresponding to amino acid positions 45-62 of SEQID NO: 8, a polypeptide molecule corresponding to amino acid positions57-74 of SEQ ID NO: 8, a polypeptide molecule corresponding to aminoacid positions 65-82 of SEQ ID NO: 8, a polypeptide moleculecorresponding to amino acid positions 111-123 of SEQ ID NO: 8, apolypeptide molecule corresponding to amino acid positions 21-38 of SEQID NO: 8, a polypeptide molecule corresponding to amino acid positions53-70 of SEQ ID NO: 8 and a polypeptide molecule corresponding to aminoacid positions 89-101 of SEQ ID NO:
 8. 8. A pharmaceutical compositionof claim 6, further comprising a polypeptide molecule selected from thegroup consisting of a polypeptide molecule corresponding to amino acidpositions 81-97 of SEQ ID NO: 8, a polypeptide molecule corresponding toamino acid positions 76-94 of SEQ ID NO: 8 and a polypeptide moleculecorresponding to amino acid positions 76-106 of SEQ ID NO:
 8. 9. Apharmaceutical composition for desensitizing a human subject to beevenom, the composition comprising: a polypeptide molecule selected fromthe group consisting of a polypeptide molecule corresponding to aminoacid positions 81-97 of SEQ ID NO: 8, a polypeptide moleculecorresponding to amino acid positions 76-94 of SEQ ID NO: 8 and apolypeptide molecule corresponding to amino acid positions 76-106 of SEQID NO: 8; and at least one pharmaceutically acceptable vehicle.
 10. Apharmaceutical composition for desensitizing a human subject to beevenom, the composition comprising: a polypeptide molecule according toSEQ ID NO: 8, wherein the molecule has at least one mutation at an aminoacid position within the range of amino acid positions 1-17, 39-44 or107-110, wherein the mutated molecule no longer exhibits reactivitytoward IgE molecules; and at least one pharmaceutically acceptablevehicle.
 11. A method of desensitizing a human subject to bee venom,comprising administering to the subject a therapeutically effectiveamount of a pharmaceutical composition of any one of claims 5, 6, 9, or10, wherein, after effective administration of said compound, thesubject is desensitized to bee venom.
 12. A method of desensitizing ahuman subject to bee venom, comprising administering to the subject atherapeutically effective amount of a pharmaceutical composition ofclaim 7, wherein, after effective administration of said compound, thesubject is desensitized to bee venom.
 13. A method of desensitizing ahuman subject to bee venom, comprising administering to the subject atherapeutically effective amount of a pharmaceutical composition ofclaim 8, wherein, after effective administration of said compound, thesubject is desensitized to bee venom.